Rapamycin analogues and the uses thereof in the treatment of neurological, proliferative, and inflammatory disorders

ABSTRACT

The present invention provides compounds of the following structure, wherein R 1 , R 2 , R 4 , R 4′ , R 6 , R 7 , and R 15  are defined above:  
                 
These compounds are useful in treating neurological disorders or complications due to stroke or head injury; benign or malignant neoplastic disease, carcinomas and adenocarcinomas; proliferative disorders; and inflammatory disorders. The compounds are therefore useful as neuroprotective and neuroregenerative, anti-proliferative, and anti-inflammatory agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/300,839, filed Dec. 15, 2005, which claims the benefit under 35 USC119(e) of prior U.S. Provisional Patent Application No. 60/637,666,filed Dec. 20, 2004.

BACKGROUND OF THE INVENTION

The present invention provides rapamycin analogues and their use in thetreatment of neurological, proliferative, and inflammatory disorders.

Ischemic stroke, which accounts for 83% of all stroke cases (theremaining 17% are of the hemorrhagic-type) occurs in approximately700,000 Americans each year, which equates to roughly 1 stroke every 45seconds. Ischemic strokes occur as a result of an obstruction within ablood vessel supplying blood to the brain. The underlying condition forthis type of obstruction is the development of fatty deposits lining thevessel walls, called atherosclerosis. These fatty deposits can cause twotypes of obstruction: 1) cerebral thrombosis, which refers to a thrombus(blood clot) that develops at the clogged part of the vessel and 2)cerebral embolism, which refers generally to a blood clot that forms atanother location in the circulatory system, usually the heart and largearteries of the upper chest and neck. A portion of the blood clot breaksloose, enters the bloodstream and travels through the brain's bloodvessels until it reaches vessels too small to let it pass. Currenttherapies to treat ischemic stroke are limited. To date, the onlyapproved drug for ischemic stroke is recombinant tissue plasminogenactivator (rt-PA). rt-PA, which acts as a thrombolytic, has a limitedtherapeutic window of opportunity (3 hours), therefore allowing only1-2% of all stroke patients to receive treatment. There are no marketedneuroprotectants agents for ischemic stroke.

Parkinson's disease (PD) is a neurodegenerative disease that isneuropathologically characterized by the selective degeneration ofdopaminergic (DAergic) neurons of the substantia nigra. PD is aprogressive disease with a mean age at onset of 55, although 15% ofpatients are diagnosed before the age of 50. It is estimated that 1.5million Americans have PD. Some of the classical signs of PD are restingtremor on one side of the body, generalized slowness of movement(bradykinesia), stiffness of limbs (rigidity), gait or balance problems(postural dysfunction). Current PD medications treat symptoms, whereasnone prevent or retard DAergic neuron degeneration.

Given their clinical importance, prototypical molecules that clearlyexhibit both neuroprotective and/or neuroregenerative activities havebeen highly sought after. Neurotrophins are a family of proteins thathave extraordinary therapeutic properties in pre-clinical models ofneurodegeneration. Although experimentally promising, clinicaldevelopment of neurotrophins was met with severe obstacles and setbacks,such as the inability to deliver these large proteins to targetpopulation of neurons, instability of the proteins, and non-specificactivity.

What is needed in the art are further compounds useful in treatingneurological, proliferative, and inflammatory disorders.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides novel compounds useful intreating, and in the preparation of medicaments useful in the treatmentof, neurological, proliferative, and inflammatory disorders.

In another aspect, the present invention provides novel neuroprotective,anti-proliferative, and anti-inflammatory agents.

In a further aspect, the present invention provides novel compoundsuseful in treating, and in the preparation of medicaments useful in thetreatment of, benign or malignant neoplastic disease, carcinomas, andadenocarcinomas.

In another aspect, the present invention provides novel compounds usefulin treating, and in the preparation of medicaments useful in thetreatment of, inflammatory disorders, including without limitation,autoimmune disorders (e.g., lupus), skin inflammatory disorders,intestinal inflammatory disorders, asthma and atopic disorders, andtransplant/graft rejection.

In yet a further aspect, the present invention provides rapamycinanalogues, and pharmaceutically acceptable salts, prodrugs, andmetabolites thereof.

In another aspect, the present invention provides methods of preparingrapamycin analogues.

In a further aspect, the present invention provides methods of treatingneurological, proliferative, cardiovascular, and inflammatory disorders.

In still another aspect, the present invention provides methods oftreating complications due to stoke or head trauma.

In yet a further aspect, the present invention provides methods oftreating benign or malignant neoplastic disease, carcinomas, andadenocarcinomas.

In still another aspect, the present invention provides methods fortreating inflammatory disorders, including without limitation,autoimmune disorders (e.g., lupus), skin inflammatory disorders,intestinal inflammatory disorders, asthma and atopic disorders, andtransplant/graft rejection.

In another aspect, the present invention provides a vascular stent orshunt which has been coated or impregnated with a compound of theinvention.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the nuclear magnetic resonance (NMR) spectra for thecompound of Example 1. The NMR spectra were obtained in d₃-acetonitrileusing a 400 MHz spectrometer.

FIG. 2 provides the NMR spectra for the compound of Example 2. The NMRspectra were obtained in d₃-acetonitrile using a 400 MHz spectrometer.

FIG. 3 provides the NMR spectra for the compound of Example 3. The NMRspectra were obtained in d₃-acetonitrile using a 400 MHz spectrometer.

FIG. 4 provides the NMR spectra for the compound of Example 4. The NMRspectra were obtained in d₃-acetonitrile using a 400 MHz spectrometer.

FIG. 5 provides the NMR spectra for the compound of Example 5. The NMRspectra were obtained in d₃-acetonitrile using a 400 MHz spectrometer.

FIG. 6 provides the NMR spectra for the compound of Example 6. The NMRspectra were obtained in d₃-acetonitrile using a 400 MHz spectrometer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel rapamycin analogues which areuseful as neuroprotective, anti-proliferative, and/or anti-inflammatoryagents. The novel rapamycin analogues have heteroatom substituents atthe 1 and 4 positions of the rapamycin backbone. In another embodiment,the present invention provides rapamycin analogues having a cyclicstructure at the 1, 2, 3 and/or 4 positions of the rapamycin backbone.

These novel compounds of the invention are useful as neuroprotectiveagents in compositions for use in treating neurological disorders. Theneurological disorder, including, e.g., a neurodegenerative orneuromuscular degenerative condition, can be a result of a geneticdisorder present at birth, a disorder developed during the lifespan ofan individual, e.g., stroke, and/or the result of physical trauma, e.g.,head, spinal injury, or injury to the peripheral nervous system.

Thus, a compound of the invention may be useful in ameliorating thesymptoms of a pre-existing neurological disorder, preventing furtherneuro- and/or neuromuscular degeneration. In some embodiments, theneuroprotective agents of the invention can be used to delay the onsetof symptoms associated with a neurological disorder.

The novel compounds of the present invention are also useful asanti-proliferative and anti-inflammatory agents and are thus useful inthe treatment of inflammatory disorders, including autoimmune disorders,arthritic disorders, skin inflammatory disorders, intestinalinflammatory disorders, asthma and atopic disorders, andtransplant/graft rejection.

It has surprisingly been found that at least two compounds of theinvention, e.g.,9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentoneand37-(4-chloro-3-methylphenyl)-9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;have anti-proliferative and immunosuppressive activity.

I. Compounds of the Invention

The present invention provides rapamycin analogues of the formula I:

R₁ and R₂ in the above-noted formula are different, independent groupsand are selected from among OR₃ and N(R_(3′))(R_(3″)) or R₁ and R₂ aredifferent, are connected through a single bond, and are selected from Oand NR₃. R₃, R_(3′), and R_(3″) are independently selected from among H,C₁ to C₆ alkyl, C₁ to C₆ substituted alkyl, C₃ to C₈ cycloalkyl,substituted C₃ to C₈ cycloalkyl, aryl, substituted aryl, heteroaryl, andsubstituted heteroaryl. R₄ and R_(4′) are (a) independently selectedfrom among H, OH, O(C₁ to C₆ alkyl), O(substituted C₁ to C₆ alkyl),O(acyl), O(aryl), O(substituted aryl), and halogen; or (b) takentogether to form a double bond to O. R₅, R₆, and R₇ are independentlyselected from among H, OH, and OCH₃. R₈ and R₉ are connected through a(i) single bond and are CH₂ or (ii) double bond and are CH. R₁₅ isselected from among C═O, CHOH, and CH₂ and n is 1 or 2; orpharmaceutically acceptable, salts, prodrugs, or metabolites thereof.

In further embodiments, R₁ and R₂ are connected through a single bondand are selected from O and NR₃. In still a further embodiment, R₁ is 0and R₂ is NR₃.

In one embodiment, R_(3′) or R_(3″) is an aryl or substituted arylgroup, or a substituted benzene ring. In another embodiment, substitutedbenzene groups at R_(3′) or R_(3″) include rings of the followingstructure:

R₁₀, R₁₁, R₁₂, R₁₃, and R₁₄ are independently selected from among H, C₁to C₆ alkyl, substituted C₁ to C₆ alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, halogen, acyl, OH, O(alkyl),O(substituted alkyl), O(aryl), O(substituted aryl), O(acyl), NH₂,NH(alkyl), NH(substituted alkyl), NH(aryl), NH(substituted aryl), andNH(acyl).

In further embodiments, R₃, R_(3′) or R_(3″) are phenyl optionallysubstituted by 1 or 2 substituents selected from C₁ to C₆ alkyl andhalogen. In still further embodiments, R₃, R_(3′) or R_(3″) are phenyloptionally substituted with 1 or 2 methyl or chloro substituents, e.g.phenyl and 3-methyl, 4-chlorophenyl.

In one embodiment, R₄ or R_(4′) are OH or O(acyl), e.g., where the acylis —C(O)— optionally substituted alkyl, in particular where alkyl can bestraight or branched and optionally substituted e.g. by heterocyclicsuch as aromatic heterocyclic such as pyridyl. An example is:

In other embodiments, rapamycin analogues of formula I include thosewhere R₅, R₆ and R₇ are OCH₃, those where the nitrogen containing ringat positions 17-22 of the rapamycin backbone is a piperidine ring, orwhere R₁₅ is a carbonyl.

In one embodiment, the invention provides compounds of the followingformula Ia:

where R₁, R₂, R₈, and R₉ are defined as noted above.

In another embodiment, the invention provides compounds of the followingformula Ib:

In formula Ib, R is independently selected from among H, C₁ to C₆ alkyl,substituted C₁ to C₆ alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, halogen, acyl, OH, O(alkyl), O(substitutedalkyl), O(aryl), O(substituted aryl), O(acyl), NH₂, NH(alkyl),NH(substituted alkyl), NH(aryl), NH(substituted aryl), and NH(acyl) andm is 1 to 5.

Specific compounds of the invention are illustrated herein and include9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl-4,9,10,12,13,14,15,16,17,18,21,22,23,24,25,26,27,32,33,34,34a-henicosahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;37-(4-chloro-3-methylphenyl)-9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;37-(2,6-dichlorophenyl)-9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentoneester with -2,2-dimethyl-3-(pyridin-2-yl)-propionic acid;37-(2,6-dichlorophenyl)-9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;or pharmaceutically acceptable, salts, prodrugs, or metabolites thereof.The invention is not limited to these illustrative compounds.

In another embodiment, the specific compounds include the following:

The present invention also provides a compound where R₁ and R₂ areconnected through a single bond; R₁ is O; R₂ is NR₃; R₃ is phenyl; R₄ isOH; R₅-R₇ are OCH₃; and R₈ and R₉ are HC═CH; a compound where R₁ is OR₃;R₂ is N(R_(3′))(R_(3″)); R₃ is H; R_(3″) is H; R_(3″) is phenyl; R₄ isOH; R₅-R₇ are OCH₃; and R₈ and R₉ are H₂C—CH₂; a compound where R₁ andR₂ are connected through a single bond; R₁ is O; R₂ is NR₃; R₃ isphenyl; R₄ is OH; R₅-R₇ are OCH₃; and R₈ and R₉ are H₂C—CH₂; a compoundwhere R₁ and R₂ are connected through a single bond; R₁ is O; R₂ is NR₃;R₄ is OH; R₅-R₇ are OCH₃; R₈ and R₉ are HC═CH; and R₃ is

a compound where R₁ and R₂ are connected through a single bond; R₁ is O;R₂ is NR₃; R₄ is OH; R₅-R₇ are OCH₃; R₈ and R₉ are HC═CH; and R₃ is

a compound where R₁ and R₂ are connected through a single bond; R₁ is O;R₂ is NR₃; R₃ is phenyl; R₅-R₇ are OCH₃; R₈ and R₉ are HC═CH; and R₄ is

and a compound where R₁ and R₂ are connected through a single bond; R₁is O; R₂ is NR₃; R₄ is OH; R₅-R₇ are OCH₃; R₈ and R₉ are H₂C—CH₂; and R₃is

The compounds of the invention can contain one or more asymmetric carbonatoms and some of the compounds can contain one or more asymmetric(chiral) centers and can thus give rise to optical isomers anddiastereomers. While shown without respect to stereochemistry, when thecompounds can contain one or more chiral centers, preferably at leastone of the chiral centers is of S-stereochemistry. Thus, the inventionincludes such optical isomers and diastereomers; as well as the racemicand resolved, enantiomerically pure stereoisomers; as well as othermixtures of the R and S stereoisomers, and pharmaceutically acceptablesalts, hydrates, metabolites, and prodrugs thereof.

The term “alkyl” is used herein to refer to both straight- andbranched-chain saturated aliphatic hydrocarbon groups having 1 to 10carbon atoms, and desirably about 1 to 8 carbon atoms. The term“alkenyl” is used herein to refer to both straight- and branched-chainalkyl groups having one or more carbon-carbon double bonds andcontaining about 2 to 10 carbon atoms. In one embodiment, the termalkenyl refers to an alkyl group having 1 or 2 carbon-carbon doublebonds and having 2 to about 6 carbon atoms. The term “alkynyl” group isused herein to refer to both straight- and branched-chain alkyl groupshaving one or more carbon-carbon triple bond and having 2 to 8 carbonatoms. In another embodiment, the term alkynyl refers to an alkyl grouphaving 1 or 2 carbon-carbon triple bonds and having 2 to 6 carbon atoms.

The term “cycloalkyl” is used herein to refer to an alkyl group aspreviously described that is cyclic in structure and has about 4 to 10carbon atoms, or about 5 to 8 carbon atoms.

The terms “substituted alkyl”, “substituted alkenyl”, and “substitutedalkynyl” refer to alkyl, alkenyl, and alkynyl groups, respectively,having one or more substituents including, without limitation, halogen,CN, OH, NO₂, amino, aryl, heterocyclic, alkoxy, aryloxy, alkylcarbonyl,alkylcarboxy, and arylthio, which groups can be optionally substitutede.g. by 1 to 4 substituents including halogen, CN, OH, NO₂, amino,alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryloxy, alkyloxy,alkylcarbonyl, alkylcarboxy, aminoalkyl, and arylthio. Thesesubstituents can be attached to any carbon of an alkyl, alkenyl, oralkynyl group provided that the attachment constitutes a stable chemicalmoiety.

The term “aryl” as used herein refers to an aromatic system, e.g., of6-20 carbon atoms, which can include a single ring or multiple aromaticrings fused or linked together (e.g. two or three) where at least onepart of the fused or linked rings forms the conjugated aromatic system.The aryl groups can include, but are not limited to, phenyl, naphthyl,biphenyl, anthryl, tetrahydtonaphthyl, phenanthryl, indene,benzonaphthyl, fluorenyl, and carbazolyl.

The term “substituted aryl” refers to an aryl group which is substitutedwith one or more substituents including halogen, CN, OH, NO₂, amino,alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryloxy, alkyloxy,alkylcarbonyl, alkylcarboxy, aminoalkyl, and arylthio, which groups canbe optionally substituted. In one embodiment, a substituted aryl groupis substituted with 1 to 4 substituents including halogen, CN, OH, NO₂,amino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryloxy, alkyloxy,alkylcarbonyl, alkylcarboxy, aminoalkyl, and arylthio.

The term “heterocyclic” as used herein refers to a stable 4- to7-membered monocyclic or multicyclic heterocyclic ring which issaturated, partially unsaturated, or wholly unsaturated, includingaromatic such as pyridyl. The heterocyclic ring has carbon atoms and oneor more heteroatoms including nitrogen, oxygen, and sulfur atoms. In oneembodiment, the heterocyclic ring has 1 to 4 heteroatoms in the backboneof the ring. When the heterocyclic ring contains nitrogen or sulfuratoms in the backbone of the ring, the nitrogen or sulfur atoms can beoxidized. The term “heterocyclic” also refers to multicyclic rings,e.g., of 9 to 20 ring members in which a heterocyclic ring is fused toan aryl ring. The heterocyclic ring can be attached to the aryl ringthrough a heteroatom or carbon atom, provided the resultant heterocyclicring structure is chemically stable.

A variety of heterocyclic groups are known in the art and include,without limitation, oxygen-containing rings, nitrogen-containing rings,sulfur-containing rings, mixed heteroatom-containing rings, fusedheteroatom containing rings, and combinations thereof. Oxygen-containingrings include, but are not limited to, furyl, tetrahydrofuranyl,pyranyl, pyronyl, and dioxinyl rings. Nitrogen-containing rings include,without limitation, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, pyridyl,piperidinyl, 2-oxopiperidinyl, pyridazinyl, pyrimidinyl, pyrazinyl,piperazinyl, azepinyl, triazinyl, pyrrolidinyl, and azepinyl rings.Sulfur-containing rings include, without limitation, thienyl anddithiolyl rings. Mixed heteroatom containing rings include, but are notlimited to, oxathiolyl, oxazolyl, thiazolyl, oxadiazolyl, oxatriazolyl,dioxazolyl, oxathiazolyl, oxathiolyl, oxazinyl, oxathiazinyl,morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, oxepinyl,thiepinyl, and diazepinyl rings. Fused heteroatom-containing ringsinclude, but are not limited to, benzofuranyl, thionapthene, indolyl,benazazolyl, purindinyl, pyranopyrrolyl, isoindazolyl, indoxazinyl,benzoxazolyl, anthranilyl, benzopyranyl, quinolinyl, isoquinolinyl,benzodiazonyl, naphthylridinyl, benzothienyl, pyridopyridinyl,benzoxazinyl, xanthenyl, acridinyl, and purinyl rings.

The term “substituted heterocyclic” as used herein refers to aheterocyclic group having one or more substituents including halogen,CN, OH, NO₂, amino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy,aryloxy, alkyloxy, alkylcarbonyl, alkylcarboxy, aminoalkyl, andarylthio, which groups can be optionally substituted. In one embodiment,a substituted heterocyclic group is substituted with 1 to 4substituents.

The term “acyl” refers to a —C(O)— group, which is substituted at thecarbon atom. The acyl group can be substituted or a terminal acyl groupsuch as an HC(O)— group. The substituents can include any substituentsnoted above for alkyl groups, viz. one or more substituents including,without limitation, halogen, CN, OH, NO₂, amino, aryl, heterocyclic,alkoxy, aryloxy, alkylcarbonyl, alkylcarboxy, and arylthio, which groupscan be optionally substituted. Examples include —C(O)-alkoxy (e.g. —OMeor —OEt) or —C(O)-alkyl where alkyl can be straight or branched andoptionally substituted e.g., by heterocyclic (such as pyridyl).

The term “alkoxy” as used herein refers to the O(alkyl) group, where thepoint of attachment is through the oxygen-atom and the alkyl group isoptionally substituted.

The term “aryloxy” as used herein refers to the O(aryl) group, where thepoint of attachment is through the oxygen-atom and the aryl group isoptionally substituted.

The term “alkyloxy” as used herein refers to the alkylOH group, wherethe point of attachment is through the alkyl group.

The term “arylthio” as used herein refers to the S(aryl) group, wherethe point of attachment is through the sulfur-atom and the aryl groupcan be optionally substituted.

The term “alkylcarbonyl” as used herein refers to the C(O)(alkyl) group,where the point of attachment is through the carbon-atom of the carbonylmoiety and the alkyl group is optionally substituted.

The term “alkylcarboxy” as used herein refers to the C(O)O(alkyl) group,where the point of attachment is through the carbon-atom of the carboxymoiety and the alkyl group is optionally substituted.

The term “aminoalkyl” as used herein refers to both secondary andtertiary amines where the point of attachment is through thenitrogen-atom and the alkyl groups are optionally substituted. The alkylgroups can be the same or different.

The term “halogen” as used herein refers to Cl, Br, F, or I groups.

II. Methods of Preparing the Compounds of the Invention

The rapamycin analogues of formula I of the present invention areprepared from a rapamycin starting material. Preferably, the rapamycinstarting material includes, without limitation, rapamycin, norrapamycin,deoxorapamycin, desmethylrapamycins, or desmethoxyrapamycin, orpharmaceutically acceptable salts, prodrugs, or metabolites thereof.However, one of skill in the art would readily be able to select asuitable rapamycin starting material that can be utilized to prepare thenovel rapamycin analogues of the present invention.

The term “desmethylrapamycin” refers to the class of rapamycin compoundswhich lack one or more methyl groups. Examples of desmethylrapamycinsthat can be used according to the present invention include3-desmethylrapamycin (U.S. Pat. No. No. 6,358,969),7-O-desmethyl-rapamycin (U.S. Pat. No. 6,399,626), 17-desmethylrapamycin(U.S. Pat. No. 6,670,168), and 32-O-desmethylrapamycin, among others.

The term “desmethoxyrapamycin” refers to the class of rapamycincompounds which lack one or more methoxy groups and includes, withoutlimitation, 32-desmethoxyrapamycin.

The rapamycin analogues of formula I of the present invention aretherefore prepared by combining a rapamycin starting material and adienophile. The term “dienophile” refers to a molecule that reacts witha 1,3-diene to give a [4+2]cycloaddition product. Preferably, thedienophile utilized in the present invention is an optionallysubstituted nitrosobenzene. A variety of nitrosobenzenes can be utilizedin the present invention and include nitrosobenzene,2,6-dichloronitrosobenzene, and 1-chloro-2-methyl-4-nitrosobenzene,among others. One of skill in the art would readily be able to selectthe amount of nitrosobenzene that would be effective in preparing therapamycin analogues of the present invention. Preferably, an excess ofthe nitrosobenzene is utilized, and more preferably in a 5:1 ratio ofnitrosobenzene to rapamycin starting material. However, even a 1:1, 2:1,or 3:1 ratio of nitrosobenzene to rapamycin can be utilized asdetermined by one of skill in the art.

The nitrosobenzene and rapamycin starting material is combined in asolvent. The solvent preferably dissolves the nitrosobenzene and/orrapamycin on contact, or dissolves the nitrosobenzene and rapamycin asthe reaction proceeds. Solvents that can be utilized in the presentinvention include, without limitation, dimethylformamide, dioxane suchas p-dioxane, chloroform, alcohols such as methanol and ethanol, ethylacetate, water, acetonitrile, tetrahydrofuran, dichloromethane, andtoluene, or combinations thereof. However, one of skill in the art wouldreadily be able to select a suitable solvent based upon the solubilityof the rapamycin starting material and nitrosobenzene, as well as thereactivity of the solvent with the same. The amount of solvent utilizeddepends upon the scale of the reaction and specifically the amount ofrapamycin starting material and nitrosobenzene present in the reactionmixture. One of skill in the art would readily be able to determine theamount of solvent required.

Typically, the solution containing the nitrosobenzene, rapamycinstarting material, and solvent is maintained at elevated temperatures,and preferably a temperature that does not promote decomposition of therapamycin and nitrosobenzene. In one embodiment, the solution ismaintained a temperature of about 30 to about 70° C., and preferablyabout 50° C. The components are heated for a period of time sufficientto permit reaction between the rapamycin and nitrosobenzene. One ofskill in the art using known techniques would readily be able to monitorthe progress of the reaction during heating and thereby determine theamount of time required to perform the reaction. In one preferredembodiment, the rapamycin and nitrosobenzene are combined with p-dioxaneand maintained at a temperature of about 50° C.

Isolation and purification of the rapamycin analogue is well within oneof skill in the art and include chromatography including, withoutlimitation, and recrystallization, high performance liquidchromatography (HPLC) such as reverse phase HPLC, and normal phase HPLC,and size-exclusion chromatography.

Once the rapamycin analogue is obtained, it can be reduced to form amore saturated rapamycin analogue. One of skill in the art would readilybe able to select a suitable reducing agent for use in the presentinvention. Preferably, reduction of the rapamycin analogue can beeffected using a hydrogenation agent. One of skill in the art wouldreadily be able to select a suitable hydrogenation agent for use in thepresent invention. Typically, transition metal catalysts or transitionmetals on a support, preferably a carbon support, among others, in thepresence hydrogen gas, are utilized to carry out the reduction. In apreferred embodiment, the reduction is performed using palladium metalon carbon in the presence of hydrogen gas.

Reduction of the rapamycin analogue is typically carried out in asolvent. A variety of solvents can be utilized in the reduction andinclude, without limitation, alcohols such as methanol. However, one ofskill in the art would readily be able to select a suitable solvent foruse in the present invention and depending on the hydrogenation catalystand rapamycin analogue being reduced. The amount of solvent depends onthe scale of the reaction, and specifically the amount of rapamycinanalogue being reduced.

The amount of hydrogenation agent utilized in the present invention canreadily be determined by one of skill in the art. However, one of skillin the art would be able to determine and adjust the amount ofhydrogenation agent necessary to perform the reduction and to form themore saturated rapamycin analogues of the present invention. Further, avariety of apparatuses can be utilized to perform the hydrogenation ofthe present invention and include Parr apparatuses, among others. Theselection of the particular apparatus for the hydrogenation is wellwithin one of skill in the art.

A preferred method of preparing the rapamycin analogues of the presentinvention is summarized in Scheme 1 below:

where R₁, R₂, R₄, R_(4′), R₆, R₇, R₁₅, and n are defined above.

The rapamycin analogues of the present invention can be utilized in theform of pharmaceutically acceptable salts, prodrugs, or metabolitesthereof derived from pharmaceutically or physiologically acceptableacids or bases. These salts include, but are not limited to, thefollowing salts with mineral or inorganic acids such as hydrochloricacid, sulfuric acid, nitric acid, phosphoric acid and organic acids suchas acetic acid, oxalic acid, succinic acid, and maleic acid. Other saltsinclude salts with alkali metals or alkaline earth metals, such assodium, potassium, calcium or magnesium in the form of esters,carbamates and other conventional “pro-drug” forms, which, whenadministered in such form, convert to the active moiety in vivo.

III. Methods of Using the Compounds of the Present Invention

The rapamycin analogues of formulae I, Ia, and Ib of the presentinvention, including the more and less saturated rapamycin analogues,are useful in applications relating to neurological disorders (includingneuromuscular disorders) and cardiovascular disorders, among others. Therapamycin analogues of formulae I, Ia, and Ib of the present invention,including the more and less saturated rapamycin analogues, are useful indisorders involving the dysfunction of calcium (Ca²⁺) ion channels, suchas ryanodine receptor (RyR1, RyR2, and Ryr3) channelopathies including,among others, malignant hyperthermia, central core disease,cathecolaminergic polymorphic ventricular tachycardia, andarrhythmogenic right ventricular dysplasia type 2 (ARVD-2). Therapamycin analogues of formulae I, Ia, and Ib of the present invention,including the more and less saturated rapamycin analogues, are alsouseful in dihydropyridine receptor channelopathies, including thoseresultant from ryanodine receptor activity due to the activity ofdihydropyridine-sensitive calcium ion (Ca²⁺) channels. As used herein,the term “channelopathy” refers to a disease or disorder involvingdysfunction of an ion channel.

The diseases and disorders referred to herein are grouped herein underconventional headings, e.g., neurological disorders and inflammatorydisorders. One of skill in the art will recognize that the diseases ordisorders referred to herein may be appropriately grouped underdifferent headings or under multiple headings. The grouping of diseasesand/or disorders referred to herein is not a limitation of the presentinvention.

The compounds of the present invention are useful in treatingneurological disorders including Alzheimer's disease; epilepsy;Huntington's Disease; Parkinson's Disease; stroke; spinal cord injury;traumatic brain injury; Lewy body dementia; Pick's disease;Niewmann-Pick disease; amyloid angiopathy; cerebral amyloid angiopathy;systemic amyloidosis; hereditary cerebral hemorrhage with amyloidosis ofthe Dutch type; inclusion body myositis; mild cognitive impairment;Down's syndrome; and neuromuscular disorders including amyotrophiclateral sclerosis (ALS), multiple sclerosis, and muscular dystrophiesincluding Duchenne dystrophy, Becker muscular dystrophy,Facioscapulohumeral (Landouzy-Dejerine) muscular dystrophy, andlimb-girdle muscular dystrophy (LGMD); and in the preparation ofmedicaments therefor. The rapamycin analogues are also useful intreating complications due to stroke, head trauma, or spinal injury, orother injuries to the brain, peripheral nervous, central nervous, orneuromuscular system, and in the preparation of medicaments therefor.

The novel rapamycin analogues are also useful as neuroprotective agents.The rapamycin analogues of the present invention may also be useful asneuroregenerative agents, i.e., restoring some neurological and/orneuromuscular or other function following onset of one of the aboveconditions and/or injury, stroke, or other trauma.

The compounds of the present invention are useful in treatingcardiovascular disorders including, but not limited to: congestive heartfailure; arrhythmogenic syndromes, including paroxysomal tachycardia,delayed afterdepolarizations, ventricular tachycardia, suddentachycardia, exercise-induced arrhythmias, long QT syndromes, andbidirectional tachycardia; thromboembolic disorders, including arterialcardiovascular thromboembolic disorders, venous cardiovascularthromboembolic disorders, and thromboembolic disorders in the chambersof the heart; atherosclerosis; restenosis; peripheral arterial disease;coronary bypass grafting surgery; carotid artery disease; arteritis;myocarditis; cardiovascular inflammation; vascular inflammation;coronary heart disease (CHD); unstable angina (UA); unstable refractoryangina; stable angina (SA); chronic stable angina; acute coronarysyndrome (ACS); first or recurrent myocardial infarction; acutemyocardial infarction (AMI); myocardial infarction; non-Q wavemyocardial infarction; non-STE myocardial infarction; coronary arterydisease; ischemic heart disease; cardiac ischemia; ischemia; ischemicsudden death; transient ischemic attack; stroke; peripheral occlusivearterial disease; venous thrombosis; deep vein thrombosis;thrombophlebitis; arterial embolism; coronary arterial thrombosis;cerebral arterial thrombosis; cerebral embolism; kidney embolism;pulmonary embolism; thrombosis resulting from (a) prosthetic valves orother implants, (b) indwelling catheters, (c) stents, (d)cardiopulmonary bypass, (e) hemodialysis, or (f) other procedures inwhich blood is exposed to an artificial surface that promotesthrombosis; thrombosis resulting from atherosclerosis, surgery orsurgical complications, prolonged immobilization, arterial fibrillation,congenital thrombophilia, cancer, diabetes, effects of medications orhormones, and complications of pregnancy; cardiac arrhytmias includingsupraventricular arrhythmias, atrial arrhythmias, atrial flutter, atrialfibrillation; other diseases listed in Heart Disease: A Textbook ofCardiovascular Medicine, 2 Volume Set, 6th Edition, 2001, EugeneBraunwald, Douglas P. Zipes, Peter Libby, Douglas D. Zipes; and in thepreparation of medicaments therefor.

In a further embodiment, the cardiovascular disease is: atherosclerosis;coronary heart disease (CHD); restensosis; peripheral arterial disease;coronary bypass grafting surgery; carotid artery disease; arteritis;myocarditis; cardiovascular inflammation; vascular inflammation;unstable angina (UA); unstable refractory angina; stable angina (SA);chronic stable angina; acute coronary syndrome (ACS); myocardialinfarction; or acute myocardial infarction (AMI), including first orrecurrent myocardial infarction, non-Q wave myocardial infarction,non-ST-segment elevation myocardial infarction and ST-segment elevationmyocardial infarction.

In still a further embodiment, the cardiovascular disease is:atherosclerosis; coronary heart disease (CHD); unstable angina (UA);unstable refractory angina; stable angina (SA); chronic stable angina;acute coronary syndrome (ACS); myocardial infarction; or acutemyocardial infarction (AMI), including first or recurrent myocardialinfarction, non-Q wave myocardial infarction, non-ST-segment elevationmyocardial infarction and ST-segment elevation myocardial infarction.

The rapamycin analogues of formulae I, Ia, and Ib of the presentinvention have also been shown to have immunosuppressive activity, andthus are useful as anti-inflammatory agents for treating inflammatorydisorders, including without limitation, autoimmune disorders (e.g.,lupus), skin inflammatory disorders, intestinal inflammatory disorders,asthma and atopic disorders, and transplant/graft rejection. Therapamycin analogues are therefore useful in the treatment ofinflammatory disorders, and in the preparation of medicaments therefor,including without limitation, autoimmune disorders, e.g., systemic lupuserythematosis (SLE), arthritic disorders (e.g., rheumatoid arthritis,psoriatic arthritis, osteoarthritis, ankylosing spondylatis), diabetesmellitus (type I), multiple sclerosis, myasthenia gravis, vasculitis;skin inflammatory disorders (e.g., psoriasis, dermatitis andscleroderma); intestinal inflammatory disorders (e.g., inflammatorybowel disease (IBD), Crohn's disease and ulcerative colitis); asthma andatopic disorders (e.g., allergy); and transplant/graft rejection andgraft v. host disease.

These compounds of formulae I, Ia, and Ib are also useful in treating orpreventing benign or malignant neoplastic disease or carcinomas andadenocarcinomas, and in the preparation of medicaments therefor. Onesuch compound is9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone.Another such compound is37-(4-chloro-3-methylphenyl)-9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone.Carcinomas and adenocarcinomas that can be treated according to thepresent invention include, without limitation, carcinomas oradenocarcinomas of the endometrium, ovary, breast, colon, renal,prostate, pituitary, meningioma or other hormone-dependent tumors.

The dosage requirements of the rapamycin analogues of the presentinvention can vary depending on the condition, severity of the symptomspresented and the particular subject being treated. One of skill in theart would readily be able to determine the amount of the rapamycinanalogue required. In one embodiment, about 0.5 to 200 mg isadministered. In a further embodiment, about 0.5 to 100 mg isadministered. In another embodiment, about 0.5 to about 75 mg isadministered. In yet a further embodiment, about 1 to about 25 mg isadministered. In another embodiment, about 0.5 to about 10 mg isadministered, particularly when used in combination with another agent.In yet a further embodiment, about 2 to about 5 mg is administered. Inyet another embodiment, about 5 to about 15 mg is administered.Treatment can be initiated with dosages of the rapamycin analoguesmaller than those required to produce a desired effect and generallyless than the optimum dose of the rapamycin analogue. Thereafter, thedosage can be increased until the optimum effect under the circumstancesis reached. Precise dosages will be determined by the administeringphysician based on experience with the individual subject being treated.In general, the compositions of this invention are most desirablyadministered at a concentration that will generally afford effectiveresults without causing any harmful or deleterious side effects.

IV. Methods of Preparing Administrable Compositions Containing theRapamycin Analogues

In one aspect, the present invention includes methods of preparing apharmaceutical composition containing one or more rapamycin analogues ofthe present invention. As used herein, reference to compositionscontaining “a rapamycin analogue” or “the rapamycin analogue” of theinvention are intended to encompass compositions containing one or morerapamycin analogues of the invention. The composition can beadministered to a mammalian subject by several different routes and isdesirably administered orally in solid or liquid form.

Solid forms, including tablets, capsules, and caplets, containing therapamycin analogue can be formed by blending the rapamycin analogue withone or more of the components described above. In one embodiment, thecomponents of the composition are dry or wet blended. In anotherembodiment, the components are dry granulated. In a further embodiment,the components are suspended or dissolved in a liquid and added to aform suitable for administration to a mammalian subject.

Liquid forms containing the rapamycin analogue can be formed bydissolving or suspending the rapamycin analogue in a liquid suitable foradministration to a mammalian subject.

Compositions containing the rapamycin analogue of the present inventioncan be prepared according to the present invention by combining therapamycin analogue and a pharmaceutically acceptable carrier.

The compositions described herein containing the rapamycin analogue canbe formulated in any form suitable for the desired route of deliveryusing a pharmaceutically effective amount of the rapamycin analogue. Forexample, the compositions of the invention can be delivered by a routesuch as oral, dermal, transdermal, intrabronchial, intranasal,intravenous, intramuscular, subcutaneous, parenteral, intraperitoneal,intranasal, vaginal, rectal, sublingual, intracranial, epidural,intratracheal, or by sustained release. Preferably, delivery is oral.

The oral dosage tablet composition of this invention can also be used tomake oral dosage tablets containing derivatives of the rapamycinanalogue, including, but not limited to, esters, carbamates, sulfates,ethers, oximes, carbonates, and the like which are known to those ofskill in the art.

A pharmaceutically effective amount of the rapamycin analogue can varydepending on the specific compound(s), mode of delivery, severity of thecondition being treated, and any other active ingredients used in thecomposition. The dosing regimen can also be adjusted to provide theoptimal therapeutic response. Several divided doses can be delivereddaily, e.g., in divided doses 2 to 4 times a day, or a single dose canbe delivered. The dose can however be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.In one embodiment, the delivery is on a daily, weekly, or monthly basis.In another embodiment, the delivery is on a daily delivery. However,daily dosages can be lowered or raised based on the periodic delivery.

The rapamycin analogues of the present invention can be combined withone or more pharmaceutically acceptable carriers or excipientsincluding, without limitation, solid and liquid carriers which arecompatible with the compositions of the present invention. Such carriersinclude adjuvants, syrups, elixirs, diluents, binders, lubricants,surfactants, granulating agents, disintegrating agents, emollients,metal chelators, pH adjustors, surfactants, fillers, disintegrants, andcombinations thereof, among others. In one embodiment, the rapamycinanalogue is combined with metal chelators, pH adjustors, surfactants,fillers, disintegrants, lubricants, and binders.

Adjuvants can include, without limitation, flavoring agents, coloringagents, preservatives, and supplemental antioxidants, which can includevitamin E, ascorbic acid, butylated hydroxytoluene (BHT) and butylatedhydroxyanisole (BHA).

Binders can include, without limitation, cellulose, methylcellulose,hydroxymethylcellulose, carboxymethylcellulose calcium,carboxymethylcellulose sodium, hydroxypropylcellulose,hydroxypropylmethylcellulose phthalate, microcrystalline cellulose,noncrystalline cellulose, polypropylpyrrolidone, polyvinylpyrrolidone(povidone, PVP), gelatin, gum arabic and acacia, polyethylene glycols,starch, sugars such as sucrose, kaolin, dextrose, and lactose,cholesterol, tragacanth, stearic acid, gelatin, casein, lecithin(phosphatides), cetostearyl alcohol, cetyl alcohol, cetyl esters wax,dextrates, dextrin, glyceryl monooleate, glyceryl monostearate, glycerylpalmitostearate, polyoxyethylene alkyl ethers, polyoxyethylene castoroil derivatives, polyoxyethylene stearates, polyvinyl alcohol, andgelatin, among others. In one embodiment, the binder is povidone,hydroxypropylmethylcellulose, carboxymethylcellulose, or gelatin. Inanother embodiment, the binder is povidone.

Lubricants can include magnesium stearate, light anhydrous silicic acid,talc, stearic acid, sodium lauryl sulfate, and sodium stearyl furamate,among others. In one embodiment, the lubricant is magnesium stearate,stearic acid, or sodium stearyl furamate. In another embodiment, thelubricant is magnesium stearate.

Granulating agents can include, without limitation, silicon dioxide,microcrystalline cellulose, starch, calcium carbonate, pectin,crospovidone, and polyplasdone, among others.

Disintegrating agents or disintegrants can include croscarmellosesodium, starch, carboxymethylcellulose, substitutedhydroxypropylcellulose, sodium bicarbonate, calcium phosphate, calciumcitrate, sodium starch glycolate, pregelatinized starch or crospovidone,among others. In one embodiment, the disintegrant is croscarmellosesodium.

Emollients can include, without limitation, stearyl alcohol, mink oil,cetyl alcohol, oleyl alcohol, isopropyl laurate, polyethylene glycol,olive oil, petroleum jelly, palmitic acid, oleic acid, and myristylmyristate.

Surfactants can include polysorbates, sorbitan esters, poloxamer, orsodium lauryl sulfate. In one embodiment, the surfactant is sodiumlauryl sulfate.

Metal chelators can include physiologically acceptable chelating agentsincluding edetic acid, malic acid, or fumaric acid. In one embodiment,the metal chelator is edetic acid.

pH adjusters can also be utilized to adjust the pH of a solutioncontaining the rapamycin analogue to about 4 to about 6. In oneembodiment, the pH of a solution containing the rapamycin analogue isadjusted to a pH of about 4.6. pH adjustors can include physiologicallyacceptable agents including citric acid, ascorbic acid, fumaric acid, ormalic acid, and salts thereof. In one embodiment, the pH adjuster iscitric acid.

Fillers that can be used according to the present invention includeanhydrous lactose, microcrystalline cellulose, mannitol, calciumphosphate, pregelatinized starch, or sucrose. In one embodiment, thefiller is anhydrous lactose. In another embodiment, the filler ismicrocrystalline cellulose.

In one embodiment, compositions containing the rapamycin analogue of theinvention are delivered orally by tablet, caplet or capsule,microcapsules, dispersible powder, granule, suspension, syrup, elixir,and aerosol. Desirably, when compositions containing the rapamycinanalogue are delivered orally, delivery is by tablets and hard- orliquid-filled capsules.

In another embodiment, the compositions containing the rapamycinanalogue can be delivered intravenously, intramuscularly,subcutaneously, parenterally and intraperitoneally in the form ofsterile injectable solutions, suspensions, dispersions, and powderswhich are fluid to the extent that easy syringe ability exits. Suchinjectable compositions are sterile and stable under conditions ofmanufacture and storage, and free of the contaminating action ofmicroorganisms such as bacteria and fungi.

In a further embodiment, compositions containing the rapamycin analoguecan be delivered rectally in the form of a conventional suppository.

In another embodiment, compositions containing the rapamycin analoguecan be delivered vaginally in the form of a conventional suppository,cream, gel, ring, or coated intrauterine device (IUD).

In another embodiment, compositions containing the rapamycin analoguecan be delivered via coating or impregnating of a supporting structure,i.e., a framework capable of containing of supporting pharmaceuticallyacceptable carrier or excipient containing a compound of the invention,e.g., vascular stents or shunts, coronary stents, peripheral stents,catheters, arterio-venous grafts, by-pass grafts, and drug deliveryballoons for use in the vasculature. In one embodiment, coatingssuitable for use include, but are not limited to, polymeric coatingscomposed of any polymeric material in which the compound of theinvention is substantially soluble. Supporting structures and coating orimpregnating methods, e.g., those described in U.S. Pat. No. 6,890,546,are known to those of skill in the art and are not a limitation of thepresent invention.

In yet another embodiment, compositions containing the rapamycinanalogue can be delivered intranasally or intrabronchially in the formof an aerosol.

The rapamycin analogues are administered orally as well as byintravenous, intramuscular, or subcutaneous routes. Solid carriersinclude starch, lactose, dicalcium phosphate, microcrystallinecellulose, sucrose and kaolin, while liquid carriers include sterilewater, polyethylene glycols, non-ionic surfactants and edible oils suchas corn, peanut and sesame oils, as are appropriate to the nature of theactive ingredient and the particular form of administration desired.Adjuvants customarily employed in the preparation of pharmaceuticalcompositions are advantageously included, such as flavoring agents,coloring agents, preserving agents, and antioxidants, for example,vitamin E, ascorbic acid, BHT and BHA.

The preferred pharmaceutical compositions from the standpoint of ease ofpreparation and administration are solid compositions, particularlytablets and hard-filled or liquid-filled capsules. Oral administrationof the compounds is preferred.

The rapamycin analogues are also administered parenterally orintraperitoneally. Solutions or suspensions of these active compounds asa free base or pharmacologically acceptable salt are prepared in watersuitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions are also prepared in glycerol, liquid, polyethylene glycolsand mixtures thereof in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form is sterile and fluid to the extentthat easy syringe ability exits. It is stable under conditions ofmanufacture and storage and is preserved against the contaminatingaction of microorganisms such as bacterial and fungi. The carrier is asolvent or dispersion medium containing, for example, water, ethanol(e.g., glycerol, propylene glycol and liquid polyethylene glycol),suitable mixtures thereof, and vegetable oil.

V. Kits of the Invention

The present invention also provides kits or packages containing therapamycin analogues. Kits of the present invention can include therapamycin analogue of the present invention and a carrier suitable foradministration to a mammalian subject as discussed above. The kits canalso contain the reagents required to prepare the rapamycin analogues ofthe present invention and include a rapamycin, an optionally substitutednitrosobenzene, and a solvent.

The kits can optionally include other reagents to form other rapamycinanalogues and include hydrogenation agents.

The kit can further contain instructions for performing the reactions ofthe present invention. Also provided in a kit can be other suitablechemicals, disposable gloves, decontamination instructions, applicatorsticks or containers, and sample preparator cups.

The following examples are provided to illustrate the invention and donot limit the scope thereof. One skilled in the art will appreciate thatalthough specific reagents and conditions are outlined in the followingexamples, modifications can be made which are meant to be encompassed bythe spirit and scope of the invention.

EXAMPLES Example 1

Table 1 provides the mass spectral (MS) data and FIG. 1 provides thenuclear magnetic resonance (NMR) spectrum for the compound produced bythe following two alternative routes. TABLE 1 Theoretical Neutral Mass:1020.59226 Exact Mass High Resolution Results Adduct Exptl. Exact mmuppm RI % [M + H]¹⁺ 1021.59780 1021.59954 −1.74 −1.70 100.0 [M + Na]¹⁺1043.57740 1043.58148 −4.08 −3.91 17.8 [M + NH₄]¹⁺ 1038.62305 1038.62608−3.03 −2.92 1.2 [M + 2H]²⁺ 511.30109 511.30341 −2.32 −4.53 1.8 [M +CH₃OH + H]¹⁺ 1053.62596 1053.62575 0.21 0.20 1.5

A. Route 1

Rapamycin (2.5 g, 2.73 mmol) was dissolved in 200 mL p-dioxane. To thissolution was added, dropwise, a solution of nitrosobenzene (1.50 g, 5eq) in 200 mL p-dioxane. The reaction mixture was stirred at 50° C. for64 hours, and then the products were chromatographed via reversed-phasehigh performance liquid chromatography (HPLC) (column: 200×50 mm YMCODS-A, mobile phase: 80% methanol:water, ramped flow rate from 10 mL/minto 35 mL/min in 10 minutes, then hold at 35 mL/min for an additional 65minutes) to yield 1.22 g of the product (44% yield, t_(R)=12.1 min,analytical HPLC conditions: column=YMC ODS-A S-3 120 Å, mobilephase/gradient: 95% water (+0.025% formic acid)/acetonitrile (+0.025%formic acid) to 5% water in 6 minutes, hold at 5% for 9 minutes,flow=0.30 mL/min).

B. Route 2—Alternate Route

Rapamycin (0.3 g, 0.328 mmol) was dissolved in 5 mL toluene with gentleheating. To this solution was added, dropwise, a solution ofnitrosobenzene (0.1 g, 3 eq) in 5 mL toluene. The reaction mixture wasstirred at 70° C. for 16 hours, and then the products werechromatographed via reversed-phase high performance liquidchromatography (HPLC) (column: 250×20 mm YMC ODS-A with 50×20 guard,mobile phase: 80 to 85% methanol:water in 40 minutes, flow=20 mL/min) toyield 0.139 g of the product (42% yield, t_(R)=12.1 min, analytical HPLCconditions: column=YMC ODS-A S-3 120 Å, mobile phase/gradient: 95% water(+0.025% formic acid)/acetonitrile (+0.025% formic acid) to 5% water in6 minutes, hold at 5% for 9 minutes, flow=0.30 mL/min).

Example 2

Table 2 provides the mass spectral (MS) data and FIG. 2 provides thenuclear magnetic resonance (NMR) spectrum for the compound produced bythe following two alternative routes.

A. Route 1

The compound prepared according to Example 1 (0.29 g, 0.284 mmol) wasdissolved in 7 mL methanol in an 18 mm test-tube, and a spatula tip ofPd/C catalyst (Aldrich) was added. The mixture was hydrogenated on aParr apparatus for 15 minutes at 2.0 atmosphere H₂. The products werechromatographed via reversed-phase HPLC (column: 250×20 mm YMC ODS-Awith 50×20 guard, mobile phase: 80% methanol:water for 15 minutes, thento 85% in 5 minutes, then held at 85% for 20 minutes, flow=20 mL/min) toyield 0.089 g of the product (31% yield, t_(R)=12.6 min, analytical HPLCconditions: column=YMC ODS-A S-3 120 Å, mobile phase/gradient: 95% water(+0.025% formic acid)/acetonitrile (+0.025% formic acid) to 5% water in6 minutes, hold at 5% for 9 minutes, flow=0.30 mL/min)

B. Route 2—Alternate Route

The compound prepared according to Example 1 (9.85 g, 9.65 mmol) wasdissolved in 50 mL methanol, and 3 spatula tips of Pd/C catalyst(Aldrich) was added. The mixture was hydrogenated on a Parr apparatusfor 2.5 hours at 2.5 atmospheres H₂. The products were chromatographedvia reversed-phase HPLC (column: 250×50 mm YMC ODS-A, mobile phase: 80%methanol:water for 40 minutes, then to 85% in 5 minutes, then held at85% for 35 minutes, flow =35 mL/min) to yield 3.35 g of the product (15%yield, t_(R)=12.2 min, analytical HPLC conditions: column=YMC ODS-A S-3120 Å, mobile phase/gradient: 95% water (+0.025% formicacid)/acetonitrile (+0.025% formic acid) to 5% water in 6 minutes, holdat 5% for 9 minutes, flow=0.30 mL/min) TABLE 2 Theoretical Neutral Mass:1022.60791 Exact Mass High Resolution Results Adduct Exptl. Exact mmuppm RI % [M + H]¹⁺ 1023.61722 1023.61519 2.03 1.99 100.0 [M + Na]¹⁺1045.59943 1045.59713 2.30 2.20 10.5

Example 3

Rapamycin (0.25 g, 0.274 mmol) was dissolved in 5 mL toluene with gentleheating. To this solution was added, dropwise, a solution of2,6-dichloronitrosobenzene (0.144 g, 3 eq) in 7 mL toluene. The reactionmixture was stirred at 80° C. for 36 hours, and then the products werechromatographed via reversed-phase HPLC (column: 250×20 mm YMC ODS-Awith 50×20 guard, mobile phase: 80 to 85% methanol:water in 40 minutes,flow=20 mL/min) to yield 0.046 g of the product (15% yield, t_(R)=13.0minutes, analytical HPLC conditions: column=YMC ODS-A S-3 120 Å, mobilephase/gradient: 95% water (+0.025% formic acid)/acetonitrile (+0.025%formic acid) to 5% water in 6 minutes, hold at 5% for 9 minutes,flow=0.30 mL/min). The MS data is provided in Table 3 and FIG. 3provides the NMR spectrum. TABLE 3 Theoretical Neutral Mass: 1088.51431Exact Mass High Resolution Results Adduct Exptl. Exact mmu ppm RI % [M +H]¹⁺ 1089.52125 1089.52159 −0.34 −0.31 19.1 [M + Na]¹⁺ 1111.500441111.50353 −3.09 −2.78 18.1 [M + NH₄]¹⁺ 1106.54443 1106.54813 −3.70−3.35 1.2

Example 4

The synthesis of this example was performed as described in Example 3and employing 0.05 g of rapamycin and 0.042 g of1-chloro-2-methyl-4-nitrosobenzene to give 0.012 g of the product (20%yield, t_(R)=12.8 min). The MS data is provided in Table 4 and FIG. 4provides the NMR spectrum. TABLE 4 Experimental Elemental FormulaPredicted Δ Δ Ion Assignment Mass (proposed) Mass (mmu) (ppm) (proposed)1091.55815 C₅₈H₈₅ClN₂O₁₄Na¹⁺ 1091.55815 0.00 0.00 [M + Na]¹⁺ 936.54361C₅₁H₇₉NO₁₃Na¹⁺ 936.54436 −0.75 −0.80 [M + Na]¹⁺—C₇H₆ClNO

Example 5

The product from Example 3 (0.046 g, 0.0422 mmol) was dissolved in 5 mLmethanol in an 18 mm test-tube, and a spatula tip of Pd/C catalyst(Aldrich) was added. The mixture was hydrogenated on a Parr apparatusfor 60 minutes at 3 atmospheres H₂. The products were chromatographedvia reversed-phase HPLC (column: 250×20 mm YMC ODS-A with 50×20 guard,mobile phase: 85% methanol:water to 90% in 15 minutes, then hold at 90%for 25 minutes, flow=20 mL/min) to yield 0.005 g of the product (11%yield, t_(R)=10.0 minutes, analytical HPLC conditions: column=YMC ODS-AS-3 120 Å, mobile phase/gradient: 95% water (+0.025% formicacid)/acetonitrile (+0.025% formic acid) to 5% water in 6 minutes, holdat 5% for 9 minutes, flow=0.30 mL/min). The MS data is provided in Table5 and FIG. 5 provides the NMR spectrum. TABLE 5 Theoretical NeutralMass: 1090.52996 Exact Mass High Resolution Results Adduct Exptl. Exactmmu ppm RI % [M + H]¹⁺ 1091.53497 1091.53724 −2.27 −2.08 4.3 [M + Na]¹⁺1113.52166 1113.51918 2.48 2.23 4.8 [M + NH₄]¹⁺ 1108.56627 1108.563782.49 2.24 15.5

Example 6

The synthesis of this example was performed as described in Example 3and using 0.108 g (0.099 mmol) rapamycin 42-ester with2,2-dimethyl-3-(pyridine-2-yl)-propionic acid (prepared according to themethod of U.S. Pat. No. 5,385,908) and 0.032 g nitrosobenzene (0.297mmol, 3 eq.). The reaction was stirred at 70 EC for 40 hours and thenthe products were chromatographed via reversed-phase HPLC (column:250×20 mm YMC ODS-A with 50×20 guard, mobile phase: 80 to 85%methanol:water in 15 minutes, then to 90% methanol in 10 minutes, thenhold at 90% for 15 minutes, flow=20 mL/min) to yield 0.007 g of theproduct (6% yield, t_(R)=13.0 minutes). The MS data is shown in Table 6and FIG. 6 provides the NMR spectrum. TABLE 6 Theoretical Neutral Mass:1181.67632 Exact Mass High Resolution Results Adduct Exptl. Exact mmuppm RI % [M + H]¹⁺ 1182.68360 1182.68747 3.87 3.28 23.2 [M + 2H]²⁺591.84544 591.84715 1.71 2.90 79.6 [M + H + Na]²⁺ 602.83641 602.837431.02 1.70 34.1

Example 7

The compound prepared according to Example 1 (0.031 g, 0.03 mmol) wasdissolved in 5 mL methanol in an 18 mm test-tube, and a spatula tip ofPd/C catalyst (Aldrich) was added. The mixture was hydrogenated on aParr apparatus for 30 minutes at 2.0 atmosphere H₂. The products werechromatographed via reversed-phase HPLC (column: 250×20 mm YMC ODS-Awith 50×20 guard, mobile phase: 80% methanol:water for 15 minutes, thento 85% in 5 minutes, then held at 85% for 20 minutes, flow=20 mL/min) toyield 0.016 g of the product (55% yield, t_(R)=9.95 min, analytical HPLCconditions: column=YMC ODS-A S-3 120 Å, mobile phase/gradient: 95% water(+0.025% formic acid)/acetonitrile (+0.025% formic acid) to 5% water in6 minutes, hold at 5% for 9 minutes, flow=0.30 mL/min) The MS data isprovided in Table 7: Exact Mass High Resolution Results Adduct Exptl.Exact mmu ppm RI % [M + H]1+ 1025.62887 1025.63084 −1.97 −1.92 38.0

Example 8

Mesencephalic dopaminergic neuron cultures were prepared as described inPong et al., J. Neurochem. 69: 986-994, 1997, which is incorporatedherein by reference in its entirety. Embryonic day 15 (E15) rat fetuseswere collected and dissected in ice-cold phosphate-buffered saline(PBS). The ventral piece of tissue compromising the mesencephalicdopaminergic region was dissected out. Dissected pieces of tissue werepooled together and transferred to an enzymatic dissociation mediumcontaining 20 IU/mL papain in Earle's balanced salt solution(Worthington Biochemical, Freehold, N.J., USA) and incubated for 60minutes at 37° C. After enzymatic dissociation, the papain solution wasaspirated and the tissue mechanically triturated with a fire-polishedglass Pasteur pipette in complete medium (equal volumes of minimumessential medium (MEM) and F-12 nutrient mixture (GibcoBRL) supplementedwith 0.1 mg/mL apotransferrin and 2.5 μg/mL insulin) containing 2,000IU/mL DNase and 10 mg/mL ovomucoid protease inhibitor.

For dopamine uptake experiments, single-cell suspensions in completemedia were seeded on poly-L-ornithine and laminin coated 24-well plates.The cultures were maintained for seven days prior to experimentation.Cultures were pretreated with various concentrations of the compound for24 hours, then exposed to 10 mM MPP+ for 1 hour. Following the 1 hourincubation, media was exchanged three times and fresh compound was addedfor an additional 48 hours.

After 48 hours growth of mesencephalic dopaminergic neuron culturesfollowing MPP+ exposure, high-affinity 3H-dopamine uptake was performedusing a modified method described by Prochiantz et al., Nature 293:570-572, 1981, which is incorporated herein by reference. Cultures werewashed with pre-warmed PBS containing 5.6 mM glucose and 1 mM ascorbicacid. Cultures were then incubated for 15 minutes at 37° C. with 50 nM3H-dopamine (31 Ci/mmol, DuPont-NEN, Wilmington, Del., USA). Thecultures were washed twice with buffer and lysed with 0.5 N NaOH. Thelysate was transferred to a scintillation vial containing Ultima Goldscintillation cocktail and radioactivity was determined with a liquidscintillation counter. Alternatively, culture lysates can be washedtwice with buffer, incubated for 2 hours at room temperature withOptiphase Supermix scintillation cocktail (Wallac ScintillationProducts, Gaithersburg, Md., USA), and radioactivity measured with aliquid scintillation counter.

Dissociated cortical neuron cultures were prepared as previouslydescribed (Pong et al., 2001). Briefly, embryonic day 15 rat fetuseswere collected and dissected in ice-cold PBS. Dissected cortices werepooled together and transferred to an enzymatic dissociation mediumcontaining papain. After 30 minutes, the tissue was mechanicallytriturated with a fire-polished glass Pasteur pipette. Single-cellsuspensions in complete media were seeded on poly-L-ornithine andlaminin coated 96-well plates. After 24 hours, cultures were treatedwith various concentrations of compound for 72 hours. The cultures werethen fixed and stained with an anti-tubulin primary antibody (TUJ-1) anda fluorescent-tagged secondary antibody. Neurite outgrowth wasdetermined by using the Enhanced Neurite Outgrowth (ENO) algorithm withthe Cellomics ArrayScan and expressed as average neurite length or totalneurite length per cell.

Spinal cord neuron cultures were prepared from embryonic day 15 (E15)rat embryos (Sprague-Dawley, Charles River Laboratories, Wilmington,Mass.). The embryos were collected and their spinal cords were removedin ice-cold phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺.Dissected pieces of spinal cord tissue were pooled together andtransferred to an enzymatic dissociation media containing 20 IU/mLpapain in Earle's balanced salt solution (Worthington Biochemical,Freehold, N.J.) and incubated for 30 minutes at 37° C. After enzymaticdissociation, the papain solution was aspirated and the tissuemechanically triturated with a fire-polished Pasteur pipette in completemedia [Neurobasal Medium with B-27 supplement (Gibco, Grand Island,N.Y.), 100 IU/mL penicillin, 100 μg/mL streptomycin, 3.3 μg/mLaphidicolin, 0.5 mM glutamate] containing 2,000 IU/mL DNase and 10-mg/mLovomucoid protease inhibitor. Single-cell suspensions in complete mediawere plated on pre-coated poly-L-omithine/laminin 96-well plates(Becton-Dickinson, Bedford, Mass.) at a density of 1.0×10⁴ cells/well.Spinal cord neurons were maintained for 24 h then exposed to vehicle orvarious concentrations of compound for 72 h.

The compounds of Examples 1-3 were all active in cortical neuron assayswith an EC₅₀ less than 1 μM. The compounds of Examples 1 and 6 were allactive in dopaminergic uptake assays with an EC₅₀ less than 1 μM. Thecompounds of Examples 1-3 and 6 were all active in spinal cord neuronassays with an EC₅₀ less than 1 μM

In comparison, CCI-779 and rapamycin were considered inactive incortical neuron assays and dopaminergic uptake assays with EC₅₀ valuesof greater than 1 μM. Rapamycin phenyltriazolinedione was active in thedopaminergic uptake assay with an EC₅₀ value of less than 1 μM.

Example 9 Permanent Occlusion of Middle Cerebral Artery (PMCAO)

Adult male Wistar rats (Charles River, Wilmington, Mass.) 270-300 g wereanesthetized with 3% isoflurane in 70% nitrous oxide and 30% oxygenthrough a nose cone. Temperature was maintained at 37° C. throughout thesurgery using a heating lamp. Permanent occlusion of MCAO was induced byelectro cauterization of the distal portion of the MCA (via acraniotomy) with a 90 min ligation of both carotid arteries to interruptcollateral circulation (Chen S T, Hsu C Y, Hogan E L, Maricq H BalentineJ D (1986) A model of focal ischemic stroke in the rat: reproducibleextensive cortical infarction. Stroke 17:738-743). Compound wasadministered 10 mg/kg i.v. 1.5, 5.5, 24, 48, and 72 hours post ischemia.Rats were kept for 21 day for long-term functional recovery evaluation.Three behavioral tests, modified from earlier tests reported by Bedersonet al., (Bederson J B, Pitts L H, Tsuji M, Nishimura M C, Davis R L,Bartkowski H (1986) Rat middle cerebral artery occlusion: evaluation ofthe model and development of a neurologic examination. Stroke 17(3):472-476) and DeRyck et al. (DeRyck M, van Reempts J, Duytschaever H, vanDeuren B, Clincke G (1992) Neocortical localization oftactile/proprioceptive limb placing reactions in the rat. Brain Res573:44-60), were used to assess sensorimotor and reflex function.Briefly, for the postural test, rats were suspended from the tailapproximately 30 cm above the bench top. Rats extending both forelimbstoward the table were scored as 0, flexing contralateral limb toward thebody and/or rotating the contralateral shoulder and limb medially werescored as 1, and rolling up the body toward the contralateral side andattempting to grasp the tail were scored as 2. The forelimb placementtest is comprised of two subtests, visual and tactile placing test. Forvisual placing test, rats were held with forelimbs hanging free and werebrought close either from front or sideway to a tabletop. For tactileplacing test, the rats were held so that it cannot see the tabletop. Thedorsal and lateral surface of the forepaw touched lightly to thetabletop. For each test, scoring was, 0 if the placing response wasimmediate and normal; 1 if the placing was delayed (>2 seconds) oroccasional; 2 if there was no response. For hind limb placement test,rats were held on the edge of the bench top and the contralateral hindlimb was pulled off the edge and released. Rats retracting hind limbback on bench top immediately were scored as 0, delaying (>2 seconds)were scored as 1, and unable to retract hind limb were score as 2. TotalScore was ranged from 0 to 12. The compound prepared in Example 2 showedstatistically significant reduction of behavior deficit scoring in ratsafter I.V. administration (10 mg/kg) following pMCAO.

Example 10 T-Cell Assays

In the following assays, human CD4+ T cells were purified by negativeselection from peripheral blood lymphocytes using RosetteSep as permanufacture's instructions (StemCell Technologies, Inc. Vancouver,British Columbia).

A. Anti-CD3/-CD28 Stimulation and IL-2 Re-stimulation Assay

Tosyl-activated magnetic microspheres (Dynal, Great Neck, N.Y.) werecoated with anti-CD3 Ab (1 μg/10⁷ microspheres), and anti-CD28Ab (0.5μg/10⁷ microspheres) as described in Blair et al. J. Immunol., 160:12,1998. Murine IgG was used to saturate the binding capacity of themicrospheres (total protein=5 μg/10⁷ microspheres). Protein-coatedmicrospheres were added to purified CD4+ T cells (2×10⁶ cells/mL, ratio1 bead: 1 cell) and activated for 72 hours in RPMI, 10% fetal calfserum, 2 mM glutamine media. Cells were harvested, washed, and culturedovernight in fresh media and re-stimulated with IL-2 as described inBennett et al., J. Immunol. 170:711, 2003. Briefly, overnight restedcells were recounted, plated (10⁵ cells/well) in flat-bottomed 96 wellmicrotiter plates and stimulated with 1 ng/mL human IL-2 (R&D Systems,Minneapolis, Minn.) in the presence of increasing concentrations ofcompound. Seventy-two hours after culture re-stimulation, plates werepulsed with 1 μCi/well tritiated thymidine and incubated for a 6-16 hourperiod.

The compounds of Examples 1 and 3 inhibited IL-2 production with IC₅₀less than about 1 μM, and preferably less than about 200 nM. The IL-2production of the compounds of Examples 1 and 3 were commensurate withthe IL-2 production of rapamycin and CCI-779.

B. Phorbol 12-myristate 13-acetate (PMA)/Ionomycin Activation Assay

Proliferation of the compounds was determined using this assay. CD4+ Tcells (5×10⁵ cells/well) were plated in flat bottomed 96 well microtiterplates in RPMI, 10% fetal calf serum, 2 mM glutamine media andstimulated with PMA (10 ng/mL) and ionomycin (200 ng/mL) in the presenceof increasing concentration of compound. Seventy-two hours afteractivation, 100 μL of culture media was collected for IL-2determination, an additional 100 μL fresh media was added to each welland cultures labeled with tritiated thymidine (1 μCi/well) for 6 hours.Cultures were harvested and tritiated thymidine incorporation (count perminute, CPM) assessed using a Trilux (Perkin Elmer, Shelton, Conn.).IL-2 production was determined using an ELISA as per manufacture'sinstructions (R&D Systems, Minneapolis, Minn.).

In this assay, the compounds of Examples 1 and 3 inhibited proliferationwith EC₅₀ ranging 4.9-0.2 μM.

Example 11

The PTEN-tumor suppressor gene (phosphate and tensin homolog deleted onchromosome ten) encodes a lipid phosphatase that plays a critical rolein the negative regulation of P13K/AKT signaling pathways. Mutation ordeletion of the PTEN gene has been found in tumors of brain, prostate,endometrium, thyroid, breast and lymphoid tumors. See, Cantley et al.,Proc. Natl. Acad. Sci. USA. 96: 4240-4245, 1999; Ali et al., J. Natl.Cancer Inst. 91: 1922-32, 1999; Scott et al., Proc. Natl. Acad. Sci.USA. 95: 7772-7777, 1998; and Nave et al., Biochem. J. 344: 427-431,1999.

In this assay, a cell-based proliferation assay was performed toevaluate the compounds of the invention in inhibiting growth of cellsincluding the breast tumor cell line MDA468 (PTEN-mutated) ATCC No.HTB-132 or prostate tumor cell line LNCap (PTEN-mutated) ATCC CRL-1740.

-   -   (i) On day one, cells were plated in 96-well culture plates.    -   (ii) On day two, compounds were added to cells at a        predetermined concentration.    -   (iii) On day five, cell proliferation was measured by standard        MTS assay protocol as described in SA O'Toole et al., Cancer        Detection and Prevention, 27(1), 2003. Results were recorded by        A490 absorbance using a 96-well format platereader.    -   (iv) The MTS results (A490 units) from compound-treated wells        were then calculated as % control growth relative to the control        (untreated) wells on the same culture plate.

This data illustrates that the compound of Example 1 had IC₅₀ values ofless than 0.01 μM for both MDA468 and LNCap cell lines.

Example 12 Modulation of Ryanodine Receptor Calcium Currents

Compounds are expected to modulate ryanodine receptor calcium currentsvia stabilization of FKBP12.6 binding to the ryanodine receptor whentested in the following assays:

A. Calcium Uptake Assay

Calcium (Ca²⁺) uptake is determined by the procedures described in (1)T. Yamamoto, et al., Cardiovascular Research, 44:146-155 (1999) or (2)M. Yano, et al., Circulation, 102:2131-2136 (2000).

Sarcoplasmic reticulum (SR) vesicles for use in these assays areprepared according to the method of Kranias, et al., Biochem. Biophys.Acta., 709:28-37 (1982). Left ventricles obtained from dogs with inducedheart failure are homogenized in a solution containing 30 mMTris-malate, 0.3 M sucrose, 5 mg/L leupeptin and 0.1 mM PMSF, at pH 7.0(Solution I). The homogenate is centrifuged at 5500 g for 10 minutes andthe resultant supernatant filtered through four layers of cheeseclothbefore centrifugation at 12,000 g for 20 minutes. The supernatant isagain filtered through cheesecloth and centrifuged at 143,000 g for 30minutes. The pellet is re-suspended in a solution containing 0.6 M KCl,30 mM Tris-malate, 0.3 M sucrose, 5 mg/L leupeptin, 0.1 mM PMSF, at pH7.0 (Solution II). The suspension is centrifuged at 143,000 g asdescribed above. The pellet is suspended in Solution I and centrifugedat 143,000 g. The pelleted microsomal fraction containing SR vesicles issuspended in a solution containing 0.1 M KCl, 20 mM Tris-malate, 0.3 Msucrose, 5 mg/L leupeptin, 0.1 mM PMSF, at pH 7.0, to give a finalprotein concentration of 10-20 mg/mL.

(1) SR vesicles as prepared above (0.2 mg/mL) are incubated in 2 mL of asolution containing 0.15 M KCl, 1 mM MgCl₂, 30 μM ⁴⁵CaCl₂ (1 mCi/mL), 10mM NaN₃ and 20 mM MOPS, pH 7.1 (22° C.). Ca²⁺ uptake is initiated byaddition of 1 mM ATP and is determined at varying time intervals byplacing a 2 mL aliquot on a 0.45 μm Millipore filter, and rinsing itwith 5 mL of washing buffer (0.15 MKCl, 20 mM MOPS, pH 7.1 (22° C.),containing 30 mM EGTA and 15 μM ruthenium red).

Radioactivity retained on the filters is determined by liquidscintillation counting.

(2) SR vesicles as prepared above (0.2 mg/mL) are incubated in 0.5 mL ofa solution containing 0.15 mol/L potassium gluconate, 1 mmol/L MgCl₂,0.2 mmol/L EGTA-calcium buffer (free [Ca²⁺] 0.3 μM), 10 mM NaN₃, and 20mM MOPS, pH 6.8. Ca²⁺ uptake is initiated by the addition of 0.5 mM ATPinto the cuvette. Ca²⁺ uptake is monitored over timespectrophotometrically with fluo 3 as a Ca²⁺ indicator (excitation at480 nm, emission at 530 nm).

B. Calcium Leak Assay Calcium (Ca²⁺) uptake is determined by theprocedures described in M. Yano, et al., Circulation, 102:2131-2136(2000).

Following a plateau in Ca²⁺ uptake in SR vesicles according to theprocedure described in “A. Calcium Uptake Assay, (2)” above, varyingconcentrations of FK506 are added in the presence of 1 μM thapsigarginto inhibit SR Ca²⁺-ATP activity, and the resultant Ca²⁺ leak ismonitored.

C. Co-Localization Assay

Co-localization of cardiac ryanodine receptor (RyR) to FKBP 12 (orFKBP12.6) is determined by the procedures described in C. George, etal., Circulation Research, 93:531-540 (2003).

HL-1 cardiomyocytes expressing the human cardiac ryanodine 2 receptors(hRyR₂) are cultured on a gelatin (0.02% [wt/vol]/fibronectin (10 μg.mL)matrix and are maintained in Claycomb media (JRH Biosciences)supplemented with fetal calf serum (10% [vol/vol], glutamine (2 mM),norepinephrine (0.1 mM), penicillin (100 u/mL), and streptomycin (100μg/mL). FKBP12.6:hRyR2 interaction is determined usingcoimmunoprecipitation assays using pAb129 (anti-RyR2) and anti-FKBP toimmunoprecipitate and immunoblot, respectively.

RyR2 is immunolocalized using pAb129 and Alexa⁴⁸⁸ conjugated secondaryantibodies and FKBP is co-stained using N-19 (Santa Cruz Biotechnology)and Alexa546 secondary antibodies.

D. Western Blots

Expression and association between cardiac ryanodine receptor (RyR) toFKBP12 (or FKBP12.6) is determined by the procedures described in C.George, et al., Circulation Research, 93:531-540 (2003).

Western blots of microsomal fractions obtained from HL-1 cardiomyocytescultured as described above in “C. Co-Localization Assay” may be carriedout using conventional techniques. Microsomal fractions (100 μg) fromHL-1 cells are immunoprecipitated using anti-RyR2 (pAb129) followed byanti-FKBP immunoblotting.

E. Electrophysiological Determination of SR Ca²⁺ Leak

Sarcoplasmic reticulum (SR) Ca²⁺ leak is determined by the proceduresdescribed in T. Shannon, et. al., Circ. Res., 93: 592-594 (2003).

Ventricular myocytes are isolated from New Zealand White rabbits(Myrtle's Rabbitry, Inc., Thompson Station, Tenn., USA) in which heartfailure is induced by combined aortic insufficiency and stenosis.Diastolic SR Ca²⁺ is measured during cellular stimulation to steadystate at different frequencies to vary load. Levels of diastolicryanodine receptor (RyR) Ca²⁺ leak may be assessed by the increasedtotal sarcoplasmic reticulum Ca²⁺ load ([Ca²⁺]SRT) upon inclusion oftetracaine (an RyR blocker).

Example 13 Cardiovascular Effect

The ability of the compounds of this invention to treat or inhibitcardiovascular disease or peripheral vascular disease is confirmed in astandard pharmacological test procedure using ApoE knockout (EKO) mice,which is a well accepted animal model of human atherosclerosis. Theprocedure used is briefly summarized below.

Male EKO mice, 4-6 weeks of age, are housed in shoe-box cages and areallowed ad lib. food and water. The animals are randomized by weightinto 5 groups (N=12-15 mice per group) and are fed Purina Rodent Chowfor the first week of the study. Also during this period as well as theremaining 12 weeks of the study, the animals are dosed every 2 days with0, 1, 2, 4 or 8 mg/kg rapamycin analogue subcutaneously (s.c.) using 2%Tween-80, 1% carboxymethyl cellulose as the vehicle and Control. Theanimal diet is switched to a casein-based Western Diet for week 2 toweek 13 of the study. At the end of the study period, the animals areeuthanized, plasma samples obtained, and the hearts perfused first withsaline, then with 10% formalin. Total cholesterol and triglycerides aredetermined using enzymatic methods with commercially-available kits fromBoehringer Mannheim and Wako Biochemicals, respectively, and theBoehringer Mannheim Hitachii 911 Analyzer 30 (Boehringer MannheimDiagnostic Laboratory Systems, Indianapolis, Ind.). Separation andquantification of plasma lipoproteins are performed using FPLC sizefractionation. Briefly, 50-100 ml of serum is filtered and injected intotwo Superose 6 columns (Amersham Pharmacia Biotech, UK, Ltd) connectedin series and eluted at a constant flow rate with 1 mM sodium EDTA and0.15 M NaCl. Areas of each curve representing VLDL, LDL and HDL areintegrated using Millennium software (Waters Technologies Corporation),and each lipoprotein fraction was quantified by multiplying the TotalCholesterol value by the relative percent area of each respective peak.The aortas are carefully isolated and remain in the formalin fixativefor 48-72 hours before handling. Atherosclerotic lesions are identifiedby Oil Red O staining, a well accepted procedure for identifyingaccumulation of neutral lipids such as cholesterols and triglycerides.The vessels are destained, and then imaged using a Nikon SMU800microscope fitted with a Sony 3CCD video camera system in concert withIMAQ Configuration Utility (National Instrument) as the image capturingsoftware. The lesions are quantified along the aortic arch using acustom threshold utility software package designed by Robert Coll(Coleman Technologies). Automated lesion assessment is performed on thevessels using the threshold function of the program, specifically on theregion contained within the aortic arch from the proximal edge of theRight Common Carotid artery to the distal edge of the Left Subclavianartery. Aortic atherosclerosis data is expressed as percent lesion(lipid) involvement strictly within this defined luminal area.Statistical significance between the Control and treated groups isdetermined using the Dunnett's Test at 1% significance level (p<0.01).

The results are anticipated to show that treatment with a compound ofthe invention significantly (p<0.0 1) increases levels of circulatingplasma HDL-cholesterol and LDL-cholesterol, while not significantlyaffecting levels of triglycerides, total cholesterol, orVLDL-cholesterol compared with control EKO mice. It is also anticipatedthat the results will show a marked and dramatic decrease in the levelof atherosclerosis (lipid deposition) in the treated mice.

The results are also expected to show that the compound of the inventionprotect against fat accumulation in the vascular wall, and thedevelopment of the classically described, atherosclerotic disease.

All patents, patent publications, and other publications listed in thisspecification are incorporated herein by reference. While the inventionhas been described with reference to a particularly preferredembodiment, it will be appreciated that modifications can be madewithout departing from the spirit of the invention. Such modificationsare intended to fall within the scope of the appended claims.

1. A compound of the formula I:

wherein: R₁ and R₂ are different, independent groups and are selectedfrom the group consisting of OR₃ and N(R_(3″))(R_(3″)); or R₁ and R₂ aredifferent, are connected through a single bond, and are selected fromthe group consisting of O and NR₃; R₃, R_(3′), and R_(3″) areindependently selected from the group consisting of H, C₁ to C₆ alkyl,C₁ to C₆ substituted alkyl, C₃ to C₈ cycloalkyl, substituted C₃ to C₈cycloalkyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; R₄ and R_(4′) are: (a) independently selected from the groupconsisting of H, OH, O(C₁ to C₆ alkyl), O(substituted C₁ to C₆ alkyl),O(acyl), O(aryl), O(substituted aryl), and halogen; or (b) takentogether to form a double bond to O; R₅, R₆, and R₇ are independentlyselected from the group consisting of H, OH, and OCH₃; R₈ and R₉ areconnected through a double bond and are CH; R₁₅ is selected from thegroup consisting of C═O, CHOH, and CH₂; n is 1 or 2; or apharmaceutically acceptable salt thereof.
 2. The compound according toclaim 1, wherein said R₁ and R₂ are connected through a single bond. 3.The compound according to claim 2, wherein R₁ is O, R₂ is NR₃.
 4. Thecompound according to claim 1, wherein R₁ is OR₃ and R₂ isN(R_(3′))(NR_(3″)).
 5. The compound according to claim 1, wherein R₃,R_(3′) or R_(3″) is an aryl or substituted aryl.
 6. The compoundaccording to claim 5, wherein said substituted aryl is a substitutedbenzene ring.
 7. The compound according to claim 6, wherein said aryl orsubstituted aryl is of the structure:

wherein: R₁₀, R₁₁, R₁₂, R₁₃, and R₁₄ are independently selected from thegroup consisting of H, C₁ to C₆ alkyl, substituted C₁ to C₆ alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, halogen, acyl, OH,O(alkyl), O(substituted alkyl), O(aryl), O(substituted aryl), O(acyl),NH₂, NH(alkyl), NH(substituted alkyl), NH(aryl), NH(substituted aryl),and NH(acyl).
 8. The compound according to claim 1, wherein R₄ or R_(4′)is OH.
 9. The compound according to claim 1, wherein R₄ or R_(4′) isO(acyl).
 10. The compound according to claim 9, wherein said acyl is:


11. The compound according to claim 1, wherein R₅, R₆, and R₇ are OCH₃.12. The compound according to claim 1, wherein n is
 2. 13. The compoundaccording to claim 1, wherein R₁₅ is C═O.
 14. The compound according toclaim 1, selected from the group selected from the group consisting of9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;37-(4-chloro-3-methylphenyl)-9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;37-(2,6-dichlorophenyl)-9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentoneester with -2,2-dimethyl-3-(pyridin-2-yl)-propionic acid; and37-(2,6-dichlorophenyl)-9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;or pharmaceutically acceptable salts of each of these.
 15. The compoundaccording to claim 1, wherein R₁ and R₂ are connected through a singlebond; R₁ is O; R₂ is NR₃; R₃ is phenyl; R₄ is OH; R₅-R₇ are OCH₃; and R₈and R₉ are HC═CH.
 16. The compound according to claim 1, wherein R₁ andR₂ are connected through a single bond; R₁ is O; R₂ is NR₃; R₄ is OH;R₅-R₇ are OCH₃; R₈ and R₉ are HC═CH; and R₃ is


17. The compound according to claim 1, wherein R₁ and R₂ are connectedthrough a single bond; R₁ is O; R₂ is NR₃; R₄ is OH; R₅-R₇ are OCH₃; R₈and R₉ are HC═CH; and R₃ is


18. The compound according to claim 1, wherein R₁ and R₂ are connectedthrough a single bond; R₁ is O; R₂ is NR₃; R₃ is phenyl; R₅-R₇ are OCH₃;R₈ and R₉ are are HC═CH; and R₄ is


19. The compound according to claim 1, which is selected from the groupconsisting of:


20. A compound of the formula Ia:

wherein: R₁ and R₂ are different, independent groups and are selectedfrom the group consisting of OH and N(R_(3′))(R_(3″)); or R₁ and R₂ aredifferent, are connected through a single bond, and are selected fromthe group consisting of O and NR₃; R_(3′) and R_(3″) are independentlyselected from the group consisting of H, C₁ to C₆ alkyl, C₁ to C₆substituted alkyl, C₃ to C₈ cycloalkyl, substituted C₃ to C₈ cycloalkyl,aryl, substituted aryl, heteroaryl, and substituted heteroaryl; R₈ andR₉ are connected through a double bond and are CH; or a pharmaceuticallyacceptable salt thereof.
 21. A compound of the formula Ib:

wherein: R is independently selected from the group consisting of H, C₁to C₆ alkyl, substituted C₁ to C₆ alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, halogen, acyl, OH, O(alkyl),O(substituted alkyl), O(aryl), O(substituted aryl), O(acyl), NH₂,NH(alkyl), NH(substituted alkyl), NH(aryl), NH(substituted aryl), andNH(acyl); and m is 1 to
 5. 22. The compound according to claim 1 whichis a neuroprotective agent.
 23. A method of treating neurologicaldisorders comprising administering to a subject in need thereof acompound as defined in claim 1 and a pharmaceutically acceptablecarrier, wherein said neurological disorder is selected from the groupconsisting of: Alzheimer's disease; epilepsy; Huntington's Disease;Parkinson's Disease; stroke; spinal cord injury; traumatic brain injury;Lewy body dementia; Pick's disease; Niewmann-Pick disease; amyloidangiopathy; cerebral amyloid angiopathy; systemic amyloidosis;hereditary cerebral hemorrhage with amyloidosis of the Dutch type;inclusion body myositis; mild cognitive impairment; Down's syndrome;amyotrophic lateral sclerosis (ALS); multiple sclerosis; Duchennedystrophy; Becker muscular dystrophy; Facioscapulohumeral(Landouzy-Dejerine) muscular dystrophy; and limb-girdle musculardystrophy (LGMD).
 24. A method of treating complications due to strokeor head trauma or treating inflammatory disorders comprisingadministering to a subject in need thereof a compound as defined inclaim 1 and a pharmaceutically acceptable carrier.
 25. The methodaccording to claim 24, wherein said inflammatory disorder is selectedfrom the group consisting of: lupus, rheumatoid arthritis, psoriaticarthritis, osteoarthritis, ankylosing spondylatis, psoriasis,dermatitis, scleroderma, inflammatory bowel disease, Crohn's disease,and ulcerative colitis.
 26. A method of treating a disorder selectedfrom the group consisting of: malignant hyperthermia; central coredisease; cathecolaminergic polymorphic ventricular tachycardia; andarrhythmogenic right ventricular dysplasia type 2 (ARVD-2); or treatinga cardiovascular disorder selected the group consisting of: congestiveheart failure; paroxysomal tachycardia; delayed afterdepolarization;ventricular tachycardia; sudden tachycardia; exercise-inducedarrhythmia; long QT syndrome; bidirectional tachycardia; arterialcardiovascular thromboembolic disorders; venous cardiovascularthromboembolic disorders; thromboembolic disorders in the chambers ofthe heart; atherosclerosis; restenosis; peripheral arterial disease;coronary bypass grafting surgery; carotid artery disease; arteritis;myocarditis; cardiovascular inflammation; vascular inflammation;coronary heart disease (CHD); unstable angina (UA); unstable refractoryangina; stable angina (SA); chronic stable angina; acute coronarysyndrome (ACS); first or recurrent myocardial infarction; acutemyocardial infarction (AMI); myocardial infarction; non-Q wavemyocardial infarction; non-STE myocardial infarction; coronary arterydisease; ischemic heart disease; cardiac ischemia; ischemia; ischemicsudden death; transient ischemic attack; stroke; peripheral occlusivearterial disease; venous thrombosis; deep vein thrombosis;thrombophlebitis; arterial embolism; coronary arterial thrombosis;cerebral arterial thrombosis; cerebral embolism; kidney embolism;pulmonary embolism; thrombosis; supraventricular arrhythmia; atrialarrhythmia; atrial flutter; and atrial fibrillation; comprisingadministering to a subject in need thereof a compound as defined inclaim 1 and a pharmaceutically acceptable carrier.
 27. A method ofpreparing a compound as defined in claim 1 comprising the steps of: (i)combining rapamycin or an analogue thereof with an optionallysubstituted nitrosobenzene; and (ii) isolating the product of (i). 28.The method according to claim 27, wherein said rapamycin analog isnorrapamycin, deoxorapamycin, or desmethylrapamycin.
 29. The methodaccording to claim 27, wherein step (i) is performed at elevatedtemperatures.
 30. The method according to claim 27, wherein step (ii) isperformed using chromatography.
 31. The method according to claim 27,for the preparation of a compound selected from the group consisting of9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;37-(4-chloro-3-methylphenyl)-9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;37-(2,6-dichlorophenyl)-9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentoneester with -2,2-dimethyl-3-(pyridin-2-yl)-propionic acid; and37-(2,6-dichlorophenyl)-9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone.32. The method according to claim 27, wherein said optionallysubstituted nitrosobenzene is selected from the group consisting ofnitrosobenzene, 2,6-dichloronitrosobenzene, and1-chloro-2-methyl-4-nitrosobenzene.
 33. The method according to claim27, further comprising: (iii) combining the product of (ii) with ahydrogenation agent; and (iv) isolating the product of (iii).
 34. Themethod according to claim 33, wherein said hydrogenation agent comprisesa Pd/C catalyst and hydrogen gas.
 35. A method of treating benign ormalignant neoplastic disease comprising administering to a subject inneed thereof a compound selected from (i)9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentoneand (ii)37-(4-chloro-3-methylphenyl)-9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;and a pharmaceutically acceptable carrier.
 36. A method of treatingcarcinomas and adenocarcinomas comprising administering to a subject inneed thereof a compound selected from (i)9,27-dihydroxy-3-{2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-37-phenyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentoneand (ii)37-(4-chloro-3-methylphenyl)-9,27-dihydroxy-3-{-2-[4-hydroxy-3-methoxycyclohexyl]-1-methylethyl}-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-4,9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-nonadecahydro-3H-23,27-epoxy-18,15-(epoxyimino)pyrido[2,1-c][1,4]oxazacyclohentriacontine-1,5,11,28,29(6H,31H)-pentone;and a pharmaceutically acceptable carrier.
 37. The method according toclaim 36, wherein said carcinomas or adenocarcinomas are of theendometrium, ovary, breast, colon, prostate, pituitary, meningioma orother hormone-dependent tumors.